The present invention relates to a vaccine for Pseudomonas aeruginosa, and in particular to novel vaccine peptides.
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Pseudomonas aeruginosa is a serious opportunistic gram-negative bacterial pathogen which can cause fatal infections in immunocompromised and immunosuppressed patients [1-4]. The first step in the infection process is the attachment to the host cell. This attachment is mediated by pili on the surface of the bacterium [2, 5, 6]. P. aeruginosa uses several adhesins to mediate attachment to mucosal surfaces, but analysis of the binding properties of the adhesins [1, 7, 8] and binding competition studies [9] indicate that the pilus is the dominant adhesin responsible for initiating infections [1].
P. aeruginosa pili are polarly located, with a structure resembling a hollow tube of 5.2 nm in outer diameter, 1.2 nm in central channel diameter, and an average length of 2.5 xcexcm [10-12]. The pilus of P. aeruginosa is composed of multiple copies of a 13-17 kDa monomeric protein subunit called pilin. The C-terminal region of the pilin monomer contains the epithelial cell binding domain [5, 12] and is semiconserved in seven different strains of this bacterium [13, 14]. This semiconserved region has also been shown to bind to a minimal structural carbohydrate receptor sequence, xcex2-GalNAc(1-4)xcex2Gal, found in glycosphingolipids, specifically asialo-GM1 and asialo-GM2 [15, 16]. Furthermore, the C-terminal disulfide-bridged 17-residue region of the PAK pilin is known to be important in raising antibodies that block binding of both bacteria or their pili to epithelial cells [6, 17, 18]. Both monoclonal antisera generated from P. aeruginosa pili or polyclonal antisera generated from synthetic peptides representing the receptor binding domain of the pathogen have been shown to be efficacious in preventing infection [19].
Different types of Pseudomonas aeruginosa immunogens have been tried or are under development as vaccines. These include lipopolysaccharides [20-22], polysaccharide [23], polysaccharide conjugate [24], outer-membrane protein [25, 26], mucoid exopolysaccharide [27], flagella [28, 29], protease [30], elastase [31], exotoxin A [31, 32], and lipoprotein I [33]. An alternate to these approaches to vaccination against P. aeruginosa could employ a multivalent pili vaccine. However, a potential problem exists in this approach: inhibition of the immune response to one antigen or determinant by the administration of another antigen or determinant. This phenomenon, termed antigenic competition [34], leads to the reduction of antibody production and has been shown to occur between chemically related and unrelated antigens and also between associated and non-associated antigenic determinants. An example of this type of competition has been reported by Hunt and coworkers [35, 36] in the development of a multivalent pili vaccine against ovine footrot. In this case, antigenic competition occurs between the nine pili serotypes of the bacterium Dichelobacter nodosus that are required in a vaccine for complete protection against the disease. These results suggested that a cocktail or multicomponent vaccine composed of synthetic peptide immunogens representing the known strains of Pseudomonas aeruginosa pili may be problematic.
The invention includes a peptide vaccine for immunizing or treating a patient for infection by a Pseudomonas aeruginosa (PA) infection. The invention comprises (i) the peptide identified as SEQ ID NOS. 3-6; and (ii) a carrier protein conjugated to the peptide.
The peptide vaccine is useful in protecting a subject against Pseudomonas infection, by administering the vaccine to the subject, also in accordance with the invention.
In another aspect, the invention includes a C-terminal PA pilin peptide having the amino acid sequence identified as SEQ ID NO: 3, and analogs thereof having one of residues T, K, or A at position 130, D, T, or N at position 132, Q, A, or V at position 133, E, P, N, or A at position 135, Q, M, or K at position 136, and I, T, L, or R at position 138, excluding SEQ ID NOS: 1, 2, 9, 10, and 11. The claimed peptide is also characterized by its ability to cross-react with antibodies against the corresponding C-terminal peptides from PA strains PAK and PAO, preferably also against antibodies specific against a C-terminal peptide from PA strains CD4, K122, or KB7.
In still another aspect, the invention includes a method of selecting a peptide for use in a vaccine against Pseudomonas aeruginosa. The method includes the steps of (i) constructing a library of 1296 C-terminal peptides having the amino acid sequence identified as SEQ ID NO: 3, and analogs thereof having one of residues T, K, or A at position 130, D, T, or N at position 132, Q, A, or V at position 133, E, P, N, or A at position 135, Q, M, or K at position 136, and I, T, L, or R at position 138, and (ii) selecting library members which are cross-reactive with cross-react with antibodies against the corresponding C-terminal peptides from PA strains PAK, PAO.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.